This invention relates to biologically active materials and the preparation thereof and, more particularly, to biologically active lymphokines and the preparation thereof from human lymphoblastoid cell line sources.
It has been shown that lymphokine-containing fractions obtained from guinea pigs exhibit many of the biological activities associated with lymphokines (D. C. Dumonde et al., Nature, 224, 38, (1969)). It has also been shown that lymphokine-containing fractions or unpurified supernatant preparations exhibit the additional capability of inducing tumor regression in vivo in man and animals (I. D. Bernstein et al., Science, 172, 729 (1971); S. B. Salvin et al., J. Natl. Cancer Inst., 55, 1233, (1975); B. W. Papermaster et al., Res. Commun. Chem. Pathol. Biol., 8, 413 (1974); B. W. Papermaster et al., Clin. Immunol. Immunopathol., 5, 31, (1975); B. W. Papermaster et al., Cancer, 45,1248, (1980); A. S. Hamblin et al., Dev. Biol. Stand., 38, 353, (1978)).
Lymphokines have been recognized since 1964 (see studies of B. R. Bloom and B. Bennet, Science, 153, 80, (1966) and J. R. David, J. Biol. Chem., 56, 72,. (1966)). The term "lymphokine" was coined by D. C. Dumonde et al. (Nature, 224, 38, (1969)). The work in the field to date has been summarized in recent international reviews and symposia (D. C. Dumonde et al., Nature, 224, 38, (1969); S. Cohen et al., Cell. Immunol., 33, 233-244, (1977); A. L. De Weck (Ed.), Biochemical Characterization of Lymphokines, Academic Press, (1980)). B. W. Papermaster et al. (Clin. Immunol. Immunopathol., 5, 31, (1976)) have described the direct effects of locally injected supernatant preparations from the human lymphoblastoid cell line, RPMI 1788, which induced histologically verified regressions in metastatic cutaneous tumor lesions. These studies were confirmed in the United Kingdom by A. S. Hamblin et al. (Dev. Biol. Stand., 38, 353, (1978) who used similar preparations for local and systemic treatment. Studies on lymphokine-induced tumor regression in the mouse have been described by S. B. Salvin et al. (J. Natl. Cancer Inst., 55, 1233, (1975)) and B. W. Papermaster et al. (Clin. Immunol. Immunopathol. 5, 31, (1976)), and the use of a lymphokine preparation in cancer patients for inflammatory skin test reactions has been described by A. Rios et al. (Cancer, 44, 1615, (1979)). Recently, L. B. Schook et al. (Biochemical Characterization of Lymphokines, A. L. De Weck (Ed.), Academic Press, (1980), p. 67) have enumerated a list of human lymphoblastoid cell lines active in lymphokine production.
Partial purification of lymphokine activities, including the Migration Inhibitory Factor (MIF) has been reported by H. G. Remold, J. Immunol., 122, 1920, (1979), lymphotoxin by M. Gately et al., Cell. Immunol., 27, 82, (1976), and others recently summarized in A. L. De Weck (Ed.), Biochemical Characterization of Lymphokines, Academic Press, (1980). Association of lymphokines with albumin and alpha-2 macroglobulin in human serum has been reported by M. C. McDaniel et al., J. Immunol. Meth., 20, 225, (1978); M. E. Smith et al., J. Immunol. Meth., 14, 243,, (1977); J. E. McEntire et al., J. Immunol. Meth., 24, 39, (1978).
It is believed that a major deficiency of the prior art is a lack of appreciation for the binding properties of active lymphokine moieties to protein carriers such as albumin and alpha-2 macroglobulin, with the attendant failure to use stringent extraction and dissociating solvents in order to obtain purified lymphokines free of carrier protein. It is also believed that another problem in the prior art is a deficiency in simple extraction procedures capable of being used with large quantities (100-1000 liters) of cell culture fluid and which do not require additional cumbersome steps to recover the extracted fractions. As a result, previous methods were capable of only producing small quantities of lymphokine preparations for laboratory use in animals or in in vitro assays, but insufficient for sustained clinical trials or biochemical characterization.
There has remained, therefore, an unfulfilled need for a more effective method for producing lymphokine fractions of a higher degree of purity and in larger quantities.